Gene targeting via homologous recombination (HR) offers a potential strategy for gene correction. Several groups have shown that gene correction at a chromosomal locus can be mediated by donor DNA fragments that are designed to be homologous to the target gene (differing only at the base pair (bp) of the mutation to be corrected) (Hu, et al., Mol. Biotech., 29:197-210 (2005); Olsen, et al., J. Gene Med., 7:1534-1544 (2005)). Other studies have shown that site-specific chromosomal damage can substantially increase the frequency of FIR by exogenous DNA (Jasin, M., Trends Genet., 12:224-228 (1996)).
One method to create site-specific DNA damage is the use of triplex-forming oligonucleotides (TFOs) which bind as third strands to homopurine/homopyrimidine sites in duplex DNA in a sequence-specific manner (Vasquez, et al., Nucleic Acids Res., 27:1176-1181 (1999)). The formation of a triple helix creates a helical distortion that has been shown to provoke DNA repair and recombination (Vasquez, et al., Proc. Natl. Acad. Sci. USA, 98:8403-8410 (2001); Wang, et al., Science, 271:802-805 (1996); Dana, et al., J. Biol. Chem., 276:18018-18023 (2001)). This approach has been used successfully to stimulate targeted recombination at chromosomal loci in mammalian cells, with recombination frequencies of up to 0.2%. Similarly, another class of DNA-binding molecules, bis-peptide nucleic acids (bis-PNAs), which can bind to homopurine regions to form PNA/DNA/PNA triplexes with a displaced DNA strand, can create PNA ‘clamps’ that also create a helical distortion that strongly provokes repair and recombination (Rogers, et al., Proc. Natl. Acad. Sci. USA, 99:16695-16700 (2002)).
However, both of these approaches are limited by the requirement for a polypurine sequence in the target duplex to enable triplex formation. To overcome this limitation, Lohse et al. Proc. Nad., Acad. Sci. USA, 96:11804-11808 (1999) reported the design of pseudo-complementary PNAs (pcPNAs), which can bind to duplex DNA at mixed purine-pyrimidine sequences via double duplex strand invasion to form four stranded complexes. To achieve pseudo-complementarity, pePNAs were synthesized with 2,6-diaminopurine (D) and 2-thiouracil (sU) nucleobases instead of As and Ts, respectively, apart from natural guanine and cytosine bases. While D and sU substitutions impede the base pairing between two mutually pseudocomplementary PNA oligomers due to steric hindrance, they do not prevent pcPNAs from binding to the corresponding sequences in DNA carrying natural nucleobases. As a result, a pair of pcPNAs can pry open a duplex DNA site via formation of double-duplex invasion complexes. This mode of pcPNA-mediated DNA recognition substantially extends the range of possible DNA targets for pcPNAs, since almost any chosen mixed-base site in duplex DNA can be targeted with pcPNAs (A+T content≦40%).
Recognition of duplex DNA at mixed sequence sites has been achieved by only two other classes of DNA binding molecules, polyamides (Home, et al., J. Am. Chem. Soc., 112:2435-2437 (1990)) and modular zinc finger polypeptides (Umov, et al, Nature, 435:646-651 (2005)). Polyamides show high affinity for duplex DNA in the minor groove, but they have not shown the ability to mediate targeted genome modification in cells. Zinc finger polypeptides, when linked to nuclease domains to form zinc finger nucleases (ZFNs), can induce recombination events in mammalian cells via the direct creation of double strand breaks, which promote recombination (Urnov, et al, Nature, 435:646-651 (2005)). Frequencies of gene modification achieved with ZFNs (plus donor DNAs) appear to be high, however, they are complex proteins that must be expressed in cells from viral or plasmid vectors, which can also produce variable levels of non-specific, off-target nuclease activity.
pcPNAs, in contrast, are relatively simple, chemically-synthesized oligomers which appear to have favorable toxicity profiles. It has been reported that pcPNAs can block access of T7 RNA polymerase to the corresponding promoter site in vitro thereby inhibiting transcription initiation (Lohse, et al., Proc. Natl. Acad. Sci. USA, 96:11804-11808 (1999)). It has also been shown that a pair of psoralen-conjugated pcPNAs can direct the formation of targeted psoralen photoadducts on duplex plasmid DNA in vitro (Kim, et al., Bioconjug. Chem., 18:567-572 (2007)) as well as at a chromosomal site in living cells, leading to the production of site-specific mutations with high efficiency and specificity (Kim, et al., Nucleic Acids, 35:7604-7613 (2007)).
To effectively correct human disease-related genes, there exists a need to improve the naturally low level of homologous recombination at chromosomal sites in human cells.
Therefore, it is an object of the invention provide recombinagenic or mutagenic compositions including a pair of pseudocomplementary oligonucleotides having sequences that form a double duplex nucleic acid molecule with a target sequence of a double-stranded nucleic acid molecule, and a donor oligonucleotide essentially complementary to a recombination target sequence of the double-stranded nucleic acid molecule, and methods for their use.
It is a further object of the invention to provide recombinagenic or mutagenic compositions having higher percentages of recombination.